The overall aim of this project is to learn more about the nature of the X chromosome fragile site (fragile X) and the factors influencing its expression in different cell types in vitro. The association between the fragile X and a major subgroup of X-linked mental retardation (MR) is now well known. However, a number of problems exist in our understanding of the fragile X, its relatinoship to MR, and its cytogenetic detection. Among these are the fact that we do not fully understand the exact biochemical or molecular nature of the fragile site or the exact relationship of the different culture variables influencing its expression. We do not know why the fragile X, when seen, is present usually in a minor porportion of cells or why it is not seen at all in cultured lymphocytes from some carrier females. We do not know if a relationship exists between the factors influencing expression of the fragile X in vitro and MR in vivo. I propose to further study the basis for expression of the fragile X in vitro by (1) determining the number of cell divisions required for expression after treatment with 5-fluorodeoxyuridine (FUdR), (2) testing the hypothesis that the fragile X can be seen in nearly all the cells in a synchronized population, (3) determining if expression is clonal in lymphoblastoid cells, and (4) determining if the need for methionine in culru medium is non-specific with regard to essential amino acids. Information gained will be used to determine optimum conditions for observing the fragile X in cultured lymphocytes, lymphoblasts and fibroblasts. I also propose to see if the fragile X can be demonstrated in fibroblasts or EBV-transformed lymphoblasts from carrier females who do not show the marker in their short-term cultured lymphocytes. Culture conditions with FUdR known to be successful with fibroblasts and lymphoblasts from affected males will be used. Reliable demonstration of the fragile X in their cultured fibroblasts or lymphoblasts would allow detection of all remales at risk for carrying the fragile X chromosome. In the 03 year of this grant, it is planned to apply analogous methodology to detect the fragile X with reliability in amniotic fluid cells.